During FY2006, we continued our exploration of the biology and evolution of the RNase A family of ribonucleases, an unusual enzyme family that is restricted to vertebrate species. While the chemistry of these enzymes has been carefully elucidated, the biological role of this highly divergent family remains for the most part unexplored. Our laboratory remains in the forefront of these biological studies, building on our past history which includes such highlights as the molecular cloning of human EDN/RNase 2 (PNAS 1989), human ECP/RNase 3 (J Exp Med 1989), elucidation of the unusual evolution of this lineage in primates (Nature Genetics 1995) and in rodents (PNAS 2000), structure function analysis of human EDN (PNAS 2002), identification and molecular cloning of human RNase 6 (NAR 1996), human RNase 8 (NAR 2002), and characterization of human EDN and ECP as antiviral ribonucleases (JID 1998; NAR 1998a, NAR 1998b). Our major publication related specifically to this project was a detailed structure-function study of chicken leukocyte RNases A-1 and A-2 (Nitto et al., JBC 2006). Separated by ~10 kb on chromosome 6, the coding sequences of RNases A-1 and A-2 are diverging under positive selection pressure (d(N) > d(S)) but remain similar to one another (81% amino acid identity) and to the mammalian angiogenins. Immunoreactive RNases A-1 and A-2 (both ~16 kDa) were detected in peripheral blood granulocytes and bone marrow. Recombinant proteins are ribonucleolytically active (k(cat) = 2.6 and 0.056 sec(-1), respectively) and, surprisingly, both interact with human placental ribonuclease inhibitor. RNase A-2, the more cationic (pI = 11.0), is both angiogenic and bactericidal; RNase A-1 (pI = 10.2) has neither activity. We demonstrated via point mutation of the catalytic His110 that ablation of ribonuclease activity has no impact on the bactericidal activity of RNase A-2. We determined that the divergent domains II (amino acids 71 - 76) and III (amino acids 89 - 104) of RNase A-2 are both important for bactericidal activity. Furthermore, we demonstrated that these cationic domains can function as independent bactericidal peptides without the tertiary structure imposed by the RNase A backbone. These results suggest that ribonucleolytic activity may not be a crucial constraint limiting the ongoing evolution of this gene family, and that the ribonuclease backbone may be merely serving as a scaffold to support the evolution of novel, non-ribonucleolytic proteins. The practical results of this study were presented to the TEAC committee in June 2006. We have been granted a provisional patent "Bactericidal peptides from avian leukocyte ribonucleases" to support the development of bactericidal RNase A-2 and encoded peptides as novel antibacterial agents. I also participated as a collaborator in exploring the results of a proteomics study carried out in the laboratory of Dr. Bin Ye of Harvard Medical School (Clin Cancer Res, 2006). The objective of the original study was to identify and characterize low molecular weight proteins/peptides in urine and their posttranslational modifications that might be used as a screening tool for ovarian cancer. Urine samples collected preoperatively from postmenopausal women with ovarian cancer and benign conditions and from nonsurgical controls were analyzed by surface-enhanced laser desorption/ ionization mass spectrometry and two-dimensional gel electrophoresis. Selected proteins from mass profiles were purified by chromatography and followed by liquid chromatography-tandem mass spectrometry sequence analysis. Specific antibodies were generated for further characterization, including immunoprecipitation and glycosylation. Quantitative and semiquantitative ELISAs were developed for preliminary validation in patients of 128 ovarian cancer, 52 benign conditions, 44 other cancers, and 188 healthy controls. A protein (m/z approximately 17,400) with higher peak intensities in cancer patients than in benign conditions and controls was identified and subsequently defined as eosinophil-derived neurotoxin (EDN). A glycosylated form of EDN was specifically elevated in ovarian cancer patients. A cluster of COOH-terminal osteopontin was identified from two-dimensional gels of urine from cancer patients. Modified forms EDN and osteopontin fragments were elevated in early-stage ovarian cancers and a combination of both resulted to 93% specificity and 72% sensitivity. We concluded that specific elevated posttranslationally modified urinary EDN and osteopontin COOH-terminal fragments in ovarian cancer might lead to potential noninvasive screening tests for early diagnosis. Urine with less complexity than serum and relatively high thermodynamic stability of peptides or metabolites is a promising study medium for discovery of the novel biomarkers which may present in many non-urinary tract neoplastic diseases. Finally, we contributed a major invited review of RNase A ribonucleases involved in host defense (Dyer & Rosenberg, Mol Diversity 2006). Covered in this review: the Ribonuclease A superfamily includes an extensive network of distinct and divergent gene lineages. Although all ribonucleases of this superfamily share invariant structural and catalytic elements and some degree of enzymatic activity, the primary sequences have diverged significantly, ostensibly to promote novel function. We reviewed the literature on the evolution and biology of the RNase A ribonuclease lineages that have been characterized specifically as involved in host defense including: (1) RNases 2 and RNases 3, also known as the eosinophil ribonucleases, which are rapidly-evolving cationic proteins released from eosinophilic leukocytes, (2) RNase 7, an anti-pathogen ribonuclease identified in human skin, and (3) RNase 5, also known as angiogenin, another rapidly-evolving ribonuclease known to promote blood vessel growth with recently-discovered antibacterial activity. Interestingly, some of the characterized anti-pathogen activities do not depend on ribonuclease activity per se. We discussed the ways in which the anti-pathogen activities characterized in vitro might translate into experimental confirmation in vivo. We also considered the possibility that other ribonucleases, such as the dimeric bovine seminal ribonuclease and the frog oocyte ribonucleases, may have host defense functions and therapeutic value that remain to be explored.